Molecular structure and tissue distribution of ryanodine receptors calcium channels

Molecular Structure and Tissue Distribution of Ryanodine Receptors Calcium Channels

Giuseppe Giannini* and Vincenzo SorrentinotSB *Department of Experimental Medicine, University of Roma; +DIBIT, San Raffaele Scientific Institute, via Olgettina

58, 20132 Milano; and #Institute of Histology, School of Medicine, University of Siena, Italy

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 11. Molecular Structure and Chromosomal Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. Potential Regulatory Regions of RyRs Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Naturally Occumng Mutations Point to the Existence of a Regulatory Domain in the

V. Expression of RyRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. RyRs in Nonmammalian Species

VII. Regulation of Calcium Release Th . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 VIII. Conclusion ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

I. INTRODUCTION

Transmission of information from the extracellular environment to the intracellular compartment is accomplished by the concerted activity of a large number of proteins. These are organized in multiple pathways, which are all connected at several steps and often function in parallel. This interaction of distinct, but interconnected, networks is required in order to guarantee the fidelity of the message that is transduced and the accuracy with which the cellular activities are regulated. Apparently, the fidelity of the system depends on the correct “wiring“ of these intracellular signaling pathways as well as on the specific growth factor, hormone or agonist that stimulates the cell. Intracellular second messengers’ job is to connect some of the proteins of the signaling machinery to one another and to other proteins which act as the distal effectors of these signals.’

Calcium ions have been established to act as second messenger in almost all eukaryotic cells, from yeast to humans, and increases in the levels of cytosolic calcium have been long acknowledged to be important in the process of intracellular signal transduction. Binding of extracellular ligands to receptors on the plasmamembrane can trigger tran- sient increases in the intracellular free calcium concentration. In most cells this is achieved by opening calcium channels located on the plasmamembrane resulting in a ”calcium influx” from extracellular fluids, or through the activation/opening of calcium channels localized on intracellular stores which leads to “calcium release” from these compartments. *

Two different families of calcium release channels have been identified which are currently known to play an important role in the regulation of calcium release from intracellular stores. The first family of calcium channels are sensitive to inositol 1, 4,5- trisphosphate (InsP,) and are referred to as the InsP, receptors (InsP,-Rs). InsP,-Rs are

§Address for correspondence: Vincenzo Sorrentino, DIBIT, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy. Tel: +39 2 2643 4726; Fax + 39 2 2643 4723.

Medicinal Research Reviews, Vol. 15, No. 4, 313-323 (1995) 0 1995 John Wiley & Sons, Inc. CCC 0198-6325/95/040313-11

314 GIANNINI AND SORRENTINO

activated by InsP, binding.2.3 Stimulation of Phospholipase C activity, as a consequence of agonist-induced stimulation of receptors on the plasmamembrane, results in increased levels InsP, and, ultimately, activates calcium release through the InsP, receptors. The second family of intracellular calcium channels is named Ryanodine receptors (RyRs) because of the ability to bind the plant alkaloid Ryanodine (Ry). The RyR family of intracellular calcium release channels will be the subject of this review. Other reviews that focus on other aspects of ryanodine receptors research have been published recently. 4-6

11. MOLECULAR STRUCTURE AND CHROMOSOMAL LOCALIZATION

Three RyRs have been identified and the relative genes are defined here as RYR1, RYR2, and RYR3. The gene for the human skeletal muscle ryanodine receptor (RYR1) was localized to the q13.1 region of chromosome 197; the cardiac human ryanodine receptor ( R Y E ) was localized on chromosome 1.8-10 The gene encoding a third RyR (RYR3) has also been recently localized and maps to the 15q14-ql5 region of the human genome." The murine homologues of these genes have also been mapped and are localized to chromosome 7A2-7A3 (RYRI), 13A1-13A2 (RYR2), and 2E5-2F3 (RYR3).12 Comparison of the regions surrounding the human and mouse RyR genes indicates that the three genes are located in evolutionary conserved linkage groups.

RyRs/calcium channels share a very similar structural organization with the InsP3-Rs. Electron microscopy and sedimentation studies have suggested a tetrameric organization for both classes of molecules, required for the formation of the real calcium gating ~hannel.5~13 This seems to occur through the interaction of several transmembrane domains located in the carboxy-terminal part of each molecule, while the remaining large amino-terminal part of the molecule is located in the cytosol.14

Cloning of the cDNA of the RyRs has provided a first idea about the structure of these large proteins. Analysis of hydropathicity profiles of the deduced aminoacid sequences has revealed that several potential transmembrane domain (TM) are present in the carboxy-terminal region of RyRs. The initial analysis performed on RyRl protein sequence has led to the proposal of two different models, based on the different require- ments used to define a transmembrane domain. Takeshima et d. suggested the existence of four transmembrane domains located in the last 500 amino acids of the rabbit RyRl

Giuseppe Giannini obtained his degree in Medicine at the University of Rome, in 1987. After an initial training in cellular immunology, he moved to study the molecular genetics of chromosomal translocation involved in human lymphomas, as a postdoctoral fellow at Raggio Italgene, Rome. In 1990 he joined Dr. Sorrentino's group at the European Molecular Biology Laboratory, in Heidelberg. In their search for new genes whose expression is regulated by TGFP, they identified a new type of Ryanodine Receptor, (RyR3. A t present he is at the Department of Pathology, at the University "La Sapienza" in Rome, where he is investigating on the molecular aspect of neuronal differentiation.

Vincenzo Sorrentino obtained his M.D. degree in 1980 at the University of Rome, Italy. In 1983 he moved to the U.S. where he worked at the NIH in Bethesda and Frederick, Ma ryland, and at the Kettering Institute for Cancer Research, Memorial Sloan Kettering, in New York City. In 1988 he joined the European Molecular Biology Laboratory in Heidelberg, Germany. Since 1993 he is Associate Professor of Histology at the School of Medicine, University of Siena and head of the "Growth Factors and Signal Transduction Laboratory" at the Department of Biology and Biotechnology of the Scientific Institute S . Raffaele in Milano, Italy. He has been working on oncogenes and growth factors, with an interest in the molecular mechanisms that control cellular proliferation. More recently he has developed an interest in intracellular calcium release channels.

RYANODINE RECEPTORS CALCIUM CHANNELS 315

protein sequence,l5 while up to 10 TM have been recognized in the last 1000 amino acids of the rabbit and human RyRl protein sequences reported by Zorzato.16 In the latter model, also sequences containing acidic residues and with mean hydropathy indices ranging from 0.8 to 1.6 were considered as putative TMs. It is conceivable, in fact, that less hydrophobic residues can also be present in complex membrane spanning regions.17 Interestingly very similar hydropathicity profiles have been observed for RyR29 and for RyR31s and our unpublished observations. The potential TM regions are also highly conserved at the protein sequence level. An exception is represented by TM3 and TM4 which are very poorly conserved between the different RYR isoforms. The presence of an even number of transmembrane domains proposed by both models mentioned above, would account for the cytosolic localization also of the short carboxy-terminal tail, as in the currently accepted model for InsP,-Rs.3

Additional information provided by protein sequence analysis contributes to the hy- pothesis that, as in the model proposed for InsP,R, a large central part of the RyR molecule is involved in modulating the calcium release activity of the three different isoforms. In fact, despite the overall high degree of conservation, at least three very divergent regions can be noticed by sequence comparison between the three RyRs. The Divergent region 1 (Dl), identified between residues 4254 and 4631 of RyRl (and corresponding residues of RyR2 and RyR3), contains the intraluminal loop between TM3 and TM4 and a bigger cytoplasmic loop between TM4 and TM5. This cytoplasmic loop also contains the calcium sensitivity domain described by Chen et al. ,19,20 see next paragraph.

The regions spanning to residues 1342-1403 (D2) and residues 1872-1923 (D3) in RyRl and the corresponding segments in RyR2 and RyR3 show significant diversity. These differences suggest that these sites may be responsible for some of the physiological and pharmacological differences between the different RyR i s ~ f o r r n s . ~ ~

Two different alternatively spliced forms have been recently described for human RyR1, both contained in the potential modulatory domain.21 The first one involves 15 bp located after G10437 in human RyRl encoding 5 amino acids (AGDIQ). In this region a difference was already noticed between the two rabbit sequences for RyRl as reported by Takeshima and Zorzato, respectively. The second alternative splicing involves the deletion of 18 bp between G11572 and G11590 in human RYRl. Interestingly enough,

RyR3 ~ ~ 1 - 1 I I I 111- --coM(

Dll D3 M

1- Pore Wming domain

-I/ //

Putam modulatory domain Putaffve IgaM brndrng doman,

Figure 1. Schematic molecular representation of the three Ryanodine Receptors. The calcium activation site and the amino acids mutated in the RyRl protein sequence of patients with MH and CCD are indicated. Transmembrane domains (open bars), divergent regions (Dl, D2, and D3). S1, S 2 splicing sites identified in human RyR1; Ser 2843 is the residue phosphorylated by PKA on Ryrl; Ser 2809 is the CaM Kinase phosphorylation site on RyR2.

316 GIA"IN1 AND SORRENTINO

small deletion at those sites can be noticed comparing the primary protein sequences for rabbit and mink RyR3, suggesting that these two alternative splicing sites could be present in at least two of the three isoforms (our unpublished observation). Unfor- tunately no experimental data are available on the functional relevance of the differentially spliced version of both RyRl and RyR3. We suppose that alternative splicing could increase the level of complexity in the regulation of the activity of these channels. As a matter of fact, alternative splicing22 involving an area near the two major sites of phosphorylation in InsP3-Rs was shown to affect the in vitro phosphorylation pattern of the differentially spliced molecules.23

111. POTENTIAL REGULATORY REGIONS OF RYRS ACTIVITY

Several agents can modify the calcium release properties of both InsP,-Rs and RyRs, such as adenine nucleotides, calcium, Mg2+ and several protein kinase.4~24 In InsP,Rs, the so-called "coupling" domain (a region located between the ligand bainding and the pore forming domains) seems to be the major target site for modulating their activity.2.3 Primary protein sequence analysis of RyRs permits the identification of several potential sites for interaction with pharmacological and physiological modulators. Takeshima et al.suggested the presence of potential calcium, ATP, and calmodulin binding sites in the sequence between residues 4253 and 4499 of the rabbit RyR1.15 Surface topology predic- tion and the analysis of protease sensitivity, as well as the identification of the major triptic fragments, suggest that this sequence is exposed on the surface of the native molecule.25 In addition, Fill et al . have indicated that polyclonal antibodies directed against the region 4445-4586, in RyRl, could reduce the open probability of the chan- ne1.26 More recently, an affinity purified antibody directed against epitopes present between residues 4478-4512 (sequence 13C2), was shown to increase the calcium sensitivity of RyRl incorporated into planar lipid bilayers, without moddying the Mg2+, ATP, Ry, and ruthenium red modulation.l9 When the 13C2 antibody was further affinity purified on the peptide sequence PEPEPEPEPE (13C2pl) included in the 13C2 fragment, it was shown, in single channel recording experiments, to specifically inhibit calcium binding and to inhibit calcium or caffeine channel activation, without affecting ATP dependent channel activity.20 It appears that the PE repeated motif is a high affinity calcium binding domain, which can be blocked by a specific antibody. When other epitopes near the PE repeat are targeted by the anti-13C2 antisera a possible local conformational change could be responsible for an increased calcium binding affinity and calcium-sensitivity.20 A different polyclonal antibody, reacting against residues 4380- 4625, which contain the PE repeat, was also shown to decrease dexorubicin and calcium- induced calcium release.27 It will be interesting to test whether the PE repeat is one of the major epitopes recognized by this antibody.

The sequence of RyRl involved in calcium sensitivity is localized in the cytoplasmic loop between TM4 and TM5, in a region very poorly conserved in RyR2 and RyR3.14 This observation suggests that calcium sensitivity domaids and other modulatory sites could be located in different regions in RyR2 and RyR3. Otsu et al. who first reported the cloning of RyR2, suggested that its main modulatory domain could map between residues 2619-3016 of their protein sequence, where potential ATP and calmodulin binding sites were identified.9 The same region also contains S2809, indicated as a potential phosphorylation site for calmodulin dependent kinase (CaM Kinase). CaM kinase, in fact, appears to preferentially phosphorylate RyR2. Direct sequencing of the phosphory-


lated triptic fragments confirmed that S2809 is indeed a relevant site for CaM kinase dependent phosphorylation.2*,29 Evidence has also been presented indicating that PKA can phosphorylate RyR2, but less efficiently than CaM kinase. However, RyR2 phosphorylation by PKA was shown to be enhanced in the presence of isoproterenol, suggesting that P-adrenergic stimulation can regulate cardiac calcium re1ease.m Serine 2843 in RyR1, corresponding to S2809 of RyR2, has been recently reported to be phosphorylated by both PKA and CaM Kinases in vitro.31c32 Evidence has been provided showing that phosphorylation can directly or indirectly affect RyRl calcium release. Patch clamp experiments, in fact, have shown that the calcium dependent inactivation of calcium release can be reversed upon phosphatase treatment or by a peptide capable of specifically inhibiting calcium/calmodulin dependent protein kinase 11, when skeletal muscle RyR was studied in its native lipid envir0nrnent.3~

Adenosine triphosphate nucleotides also seem to affect RyRs activity, and several potential sites for ATP binding are present on all isoforms. Several potential binding sites for calmodulin, another regulator of calcium release from both RyRl and RyR2, have been described on both receptors. A potential calmodulin binding site has been identified at residues 3472-3495 of the rabbit brain RyR318 and is conserved in RyRl and RyR2.

IV. NATURALLY OCCURRING MUTATIONS POINT TO THE EXISTENCE OF A REGULATORY DOMAIN IN THE N-TERMINAL REGION OF RYRS

Experimental data have indicated that the very N-terminal800 amino acids of InsP,-Rs are involved in InsP, binding. It has been suggested that InsP, binding can cause a molecular conformational change leading to channel opening. It is possible that the N-terminal region is directly involved in ligand binding and channel activation also in RyR molecules.3 This idea is supported by the localization of a point mutation involved in the development of Malignant Hyperthermia both in humans and pigs.34~35 Malignant hyperthermia susceptibility (MHS) is an autosomal dominant disorder of the skeletal muscle that manifests as a hypermetabolic crisis, commonly observed in genetically predisposed humans following inhalation of anaesthetics and exposure to depolarizing muscle relax- ants.% Alothane and succinylcholine can in fact induce, with a frequency of about 1 in 15,000 children or 1 in 50,000 adults, skeletal muscle rigidity followed by hypermetabol- ism, increased body temperature, and, if untreated, death. MH in humans has been ascribed to a defect in the calcium release channel of skeletal muscle Sarcoplasmic Reticulum (SR). In SR, calcium release from the SR of individuals affected by MH, can be induced with lower concentration of calcium, adenosine trisphosphate, and caffeine compared to normal controls. The skeletal muscle ryanodine receptor gene (RYR 1) has been shown to be linked to the locus for Malignant Hyperthermia Suscep- tibility (MHS) in at least a subset of the MHS families studied.7,37 A similar disease has also been observed in pigs and linkage analysis has also implicated the RYRl in swine MHS. A single point mutation, changing a C to a T at nucleotide 1843 in the RYRl gene and thus substituting a Cys for Arg615, has been identified as the only possible cause of Malignant Hyperthermia (MH) in swine.35 Are15 has also been linked to some MH families, in humans.34 Arg615 and the surrounding protein sequence is strongly conserved between species and between the different isoforms, suggesting a physiological relevance for this sequence. Other mutations have also been identified in the RyRl cDNA from patients affected by MH and/or ”central core disease,” another inherited myopathy that has been


associated with predisposition to MH and has been mapped to chromosome 19q13.21,38 These mutations are: GlyZaArg, Arg23=His, Arg'mCys, and IleN3Met. With the only exception of Arg2334His, all the mutations linked so far to these human pathologies appear to be clustered in a very small region, between residues 163 and 615 of the human RyRl sequence, immediately upstream from the four repeated sequences (occurring in two tandem pairs) which are maintained in all three isoforms. All the mutated residues, excepted for Gly248, appear to be conserved in the three isoforms and in different species and are contained in areas with considerable sequence conservation.

V. EXPRESSION OF RYRS

For a long time, calcium release through RyRs has been almost of exclusive interest of muscle physiologists, mainly because of the initial assumption that their expression was restricted to muscle fibers. However, in the past years pharmacological studies have indicated that it was possible to obtain calcium release from intracellular stores upon treatment with caffeine or ryanodine of several noncontractile cell types such as neurons, cromaffin cells, and insulin secreting cells.' Furthermore, new data, coming from a more extensive use of [3H]-ryanodine binding experiments, antibodies, and nucleic acid probes, have provided evidence for RyRs being expressed and functional also in non- muscle cells.4~6,~4

The skeletal muscle specific RyR (RYR1) was found to be expressed in fast- and slow- twitch muscle,15,16,39 and a second, distinct subtype, in the heart.9.40 In mammals, studies on muscle development suggested that these same isoforms are expressed in skeletal and cardiac muscles during ontogenesis and adult life.41

Although at much lower levels, compared to RyRl, RyR3 is also shown to be expressed in mammalian skeletal muscle.42 It appears thus that two different RyRs can be expressed in skeletal muscle. Whether RyRl and RyR3 are coexpressed in the same muscle fiber or whether different fiber types can express different RyR subtypes still remains to be demonstrated. RyR3 mRNA was also detected by RNase protection and in situ hybridization in the heart,42 where it seems to be selectively expressed in the con- ductive tissue, together with InsP3-Rs.43

Calcium release from intracellular stores seems to be involved in the control of various neural activities such as cell excitability, neurotransmitter release, long-term poitentiation, gene expression, and differentiation.1.44 InsP,, Ry, and caffeine-sensitive pools have been demonstrated in peripheral and central neurons. A differential distribution of InsP, and caffeine-sensitive calcium store has been detected in rat brain.45146 RyRs appear to be more expressed at the level of the pyramidal cell of CA3 and the molecular cell layer of the dentate gyrus, in the hippocampus, while InsP3-Rs are particularly enriched in CAI pyramidal cells. A differential expression of RYR and InsP3-Rs has also been found in the cortical region, in the olfactory bulb, in the cerebellum, and in the corpus striatum.47-49

A RyR has also been purified from rabbit brain:49 it shows similarity with the cardiac type 2 isoform. This could imply that the RyR2 isoform may be the major subtype of RyR expressed in the brain. However, other ryanodine receptors genes, namely RYRl and RYR3, are expressed in the brain. A rabbit homologue of RYR3 has been cloned from the brain and has been found by Northern hybridization to be expressed mainly in the corpus striatum, thalamus, and hippocampus.'s Immunoblot analysis and immu- nohistochemistry using specific antibodies demonstrated that RyRl is specifically restricted to Purkinje cells in cerebellum.50


We have performed a detailed characterization of the distribution of the three RyRs mRNAs in mouse brain by RNase protection and in situ hybridization analysis and found that all three different RYR mRNAs are expressed in mouse forebrain and cerebellum.51 In the cerebellum, RyRl expression is restricted to the Purkinje cells, while RyR2 is strongly expressed in the granular cell layer where also RyR3 is expressed, although at lower levels. A differential expression was also demonstrated in the hippocampus: RyRl probe prevalently stains the dentate gyrus and at lower levels CA1 pyramidal cells in the Ammon’s horn; RyR2 is abundantly expressed in the whole structure, with higher levels in the dentate gyrus and in CA1 pyramidal cells. The signal for RyR3 appears to be stronger in CA1 and lighter in CA3 and in the dentate gyrus. Caudate/putamen nuclei are strongly labeled by the RyR3 probe and, at lower levels by the one for RyRl. RyR2 expression, on the contrary, was detected at the level of the cortex, in the lateral septum, in the medial habenula nuclei, and in the amygdala. RyR3 was also detected in the thalamic and hypothalamic area. RyR3 signal was also observed in the region were the dorsalis raphae nuclei are located.

In conclusion RyRs appear to be widely expressed throughout the mouse central nervous system, with a pattern which seems to be often complementary. In the hippocampus, more than one receptor is found in the same cell layer. It is likely that in many cases more than one channel can be expressed in the same cell type, suggesting that the functional activities mediated by each molecule could be different.

Expression of RyRs has also been described in peripheral tissues.42 In fact, mink RyR3 was found to be expressed in skeletal muscle, jejunum, ileum, kidney, lung, stomach, and spleen, as detected by RNase protection. Rabbit RyR3 was also shown to be present in smooth muscle containing tissues like aorta, esophagus, taenia coli, urinary bladder, ureter, and uterus.18 Expression of RyR2 was documented in the stomach4 and in vascular and endocardia1 endothelium.52

The analysis of a large panel of mouse tissues, examined by RNase protection for the expression of the three isoforms of RyRs also support the idea of a broader pattern of expression for these molecules.51 In fact, a RyRl probe detects the corresponding transcript in several tissues, such as esophagus, spleen, gut, stomach, submaxillary gland, thymus, testis, adrenal gland, and ovary. Also RyR2 seems to be expressed in several tissues, among which lung, esophagus, gut, stomach, thymus, adrenal gland, and ovary. In agreement with the results described for mink tissues, RyR3 expressed in mouse lung, esophagus, spleen, gut, kidney, stomach, submaxillary gland, testis, adrenal glands, and ovary.

RyRl and RyR3 were also detected in mouse testis by RNase protection. In situ hybridization has confirmed this data, indicating that mRNAs for both molecules are detected in germinal cells, including both spermatides and spermatocytes, but not in the Sertoli cells.51 The functional significance of this finding is not known, but we can speculate that also in male germinal cells, RyRs are involved in calcium signaling and that their function is probably restricted to a specific stage of cell differentiation.

VI. RYRS IN NONMAMMALIAN SPECIES

Muscle type RyRs have also been studied in avian, where three different subtypes have been described. Expression of a cardiac specific subtype, similar to the mammalian RyR2 protein, is observed in chicken heart and cerebellum, while two different subtypes, a and p seem to be expressed in skeletal muscle. They both appear to be bio-


chemically and immunologically different from the RyRs expressed in heart. A similar pattern of expression has been observed in frogs and fish.

In chicken, a and P subtypes are differently expressed during muscle development, with the a subtype being expressed at day 10 and P subtype first appearing at day 15 of embryonic development. Failure to express the ci subtype appears to be tightly associated with the croocked neck Dwarf (cn) mutation in chicken. No substantial variation in the expression of the P-subtype was found between control and on mutant chicken.53,-

Ry binding proteins have been identified in chicken brain extract, and different proteins, recognized by a monoclonal antibody developed against avian skeletal muscle RyR, were detected in avian central nervous system.55 In Purkinje cells they appear to be coexpressed with InsP3-Rs.56 Recently, two different genes have been cloned from a chicken skeletal muscle cDNA library. Computer-aided analysis of these two nucleotide sequences indicates that they are the chicken homologues of mammalian RYRl and RYR3, respectively. Specific antisera developed against the chicken homologues of RyRl and RyR3 were able to recognize, respectively, the previously described ci and P subtype, when used to probe western blots from skeletal muscle microsome preparations (Ottini and Sorrentino, submitted for publication). A similar conclusion has been reached also for the frog a and P subtypes that have been recently shown to correspond to the RYRl and RYR3 genes.57

Calcium release through RyRs was also shown to play a role in sea urchin eggs. Caffeine, Ry, and cADPR sensitive store have been involved, together with InsP3 sensitive store, in fertilization-induced calcium release.58 Anti-skeletal muscle RyR antibodies showed positive immunoflorescence on sea urchin eggs, with most of the signal localized in the cortex.59

A cDNA for the Drosophila homologue of RyRs has been cloned.60 Expression of RyR genes has also been studied in adult Drosophila and at different times during development. Transcripts of the gene are expressed in the mesoderm of early stage-9 Drosophila embryos and later on in somatic muscles. In adult flies, the RyR mRNA is expressed in tubular muscles and neuronal tissues.61 RyRs have also been studied in lobster and in C. EIegans.62,63 Single-channel analysis of these receptors shows functional properties com- parable to those of mammals but also indicates differences in the degree of sensitivity to calcium and to antagonist of channel activity.

VII. REGULATION OF CALCIUM RELEASE THROUGH RYRS

The regulatory pathways and the mechanisms that activate calcium release through these channels have been the object of several studies and new information has been obtained. It has been proposed that, in skeletal muscle, RyRl may be opened through a direct interaction with the skeletal dihydropyridine receptor (DHPR) located on the plasmamembrane. This process does not require the influx of calcium ions. On the contrary, in cardiac muscle, activation of RyR2 by the cardiac dihydropyridine receptor requires an influx of calcium.

Several modulators which can regulate the channel function of RyRs have been identified. Calcium and ATP act as channel agonists, while magnesium and ruthenium red can inhibit channel activity.* Calcium remains a strong candidate for regulating calcium flux through RyRs, through the so-called calcium-induced-calcium-release (CIRC) mechanisms. This mechanism may also link the opening of RyRs, via intracellular calcium arelease, with activation of InsP3-Rs. * Cyclic ADP-ribose (cADPR), a novel NAD+ metab- olite, is the strongest candidate for a role as RyRs activator.58,@,= cADPR is a potent


calcium-mobilizing agent which acts on a calcium-release mechanism different from the one activated by InsP,, and recent work suggests that it modulates a Ry-sensitive, but InsP,-insensitive, calcium channel of the endoplasmic reticulum.&-@ cADPR is widely present in mammalian tissues at concentrations ranging between 20 and 100 nM.&,67,70

Many studies have been carried out with sea urchin egg microsomes, the initial system that proved the existence of cADPR.68 In sea urchin egg microsomes cADPR releases calcium with a half-maximal response at 18 nM, significantly lower than that for InsP,. cADPR binds to a specific site on sea urchin egg microsomes with a KD of 17 nM and a BmaL of 25 fmol mg-1,6*,71 Studies on cADPR-induced calcium-release are consistent with an activity of this compound on RyRs. Treatment with agonists of the RyRs, like caffeine, Ry and calcium abolished the subsequent effect of cADPR, but not of InsP, on calcium release. Conversely, blockers of RyRs, such as ruthenium red and procaine, abrogated caffeine-, Ry-, and cADPR-induced calcium release but did not affect InsP, activity. cADPR binding is not competed by the In#,-Rs antagonist heparin, suggesting that the two binding sites are distinct.

Although cADPR has all the characteristics necessary as an important candidate for an endogenous ligand of the RyRs, more evidences are still required in order to prove that cADPR is really a second messenger and does not function as a modulator of the activity of these channels.

Independently of the exact role of cADPR on RyRs activation, it is clear that these large molecules are very sophisticated in their regulation and that the different isoforms differ in their sensitivity to the different agonists. The skeletal RyR is activated, in skeletal muscle, by some conformational change induced by the DHPR.5.6 It can also be activated by calcium, but it is poorly sensitive to the calcium-releasing effect of cADPR.7'2 Calcium can of course regulate the RyR type 2, which is also highly sensitive to cADPR.73

VIII. CONCLUSION

RyRs function in regulating intracellular calcium levels in skeletal and cardiac muscle has been recognized for a long time. More recently the presence, and thus a role of RyRs in calcium signaling in other cell types has also been reported. Expression and function of these receptors in brain is now well documented. Furthermore, the presence of these channels has been reported both by pharmacological and molecular techniques, in many, if not all, tissues studied. Interestingly, this widespread pattern originally reported only for the RYR3 gene, has also been extended to RYRl and RYR2. We expect to see in the next years several advances in the field of RyRs. This will help to better under- stand their contribution to the regulation of calcium homeostasis and to the intracellular signaling in the cell types where they are present.

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Molecular structure and tissue distribution of ryanodine receptors calcium channels

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